The Journal of Neurophysiology Vol. 82 No. 1 July 1999, pp. 10-15
Copyright ©1999 by the American Physiological Society

Alphaxalone Activates a Cl Conductance Independent of GABA_A Receptors in Cultured Embryonic Human Dorsal Root Ganglion Neurons

 ¹Neurophysiology and Spinal Cord Pharmacology Laboratories, Veterans Affairs Medical Center; and  ²Department of Neurology and  ³The Miami Project to Cure Paralysis, University of Miami School of Medicine, Miami, Florida 33101

Valeyev, Alexander Y., John C. Hackman, Alice M. Holohean, Patrick M. Wood, Jennifer L. Katz, and Robert A. Davidoff. Alphaxalone Activates a Cl Conductance Independent of GABA_A Receptors in Cultured Embryonic Human Dorsal Root Ganglion Neurons. J. Neurophysiol. 82: 10-15, 1999.Whole cell and cell-attached patch-clamp techniques characterized the neurosteroid anesthetic alphaxalone's (5-pregnane-3-ol-11,20-dione) effects on GABA_A receptors and on Cl currents in cultured embryonic (5- to 8-wk old) human dorsal root ganglion neurons. Alphaxalone applied by pressure pulses from closely positioned micropipettes failed to potentiate the inward Cl currents produced by application of GABA. In the absence of GABA, alphaxalone (0.1-5.0 µM) directly evoked inward currents in all dorsal root ganglion neurons voltage-clamped at negative membrane potentials. The amplitude of the current was directly proportional to the concentration of alphaxalone (Hill coefficient 1.3 ± 0.15). The alphaxalone-induced whole cell current was carried largely by Cl ions. Its reversal potential was close to the theoretical Cl equilibrium potential, changing with a shift in the external Cl concentration as predicted by the Nernst equation for Cl ions. And because the alphaxalone-current was not suppressed by the competitive GABA_A receptor antagonist bicuculline or by the channel blockers picrotoxin and t-butylbicyclophosphorothionate (TBPS; all at 100 µM), it did not appear to result from activation of GABA_A receptors. In contrast to GABA-currents in the same neurons, the whole cell current-voltage curves produced in the presence of alphaxalone demonstrated strong inward rectification with nearly symmetrical bath and pipette Cl concentrations. Fluctuation analysis of the membrane current variance produced by 1.0 µM alphaxalone showed that the power density spectra were best fitted to double Lorentzian functions. The elementary conductance for alphaxalone-activated Cl channels determined by the relationship between mean amplitude of whole cell current and variance was 30 pS. Single-channel currents in cell-attached patches when the pipette solution contained 10 µM alphaxalone revealed a single conductance state with a chord conductance of ~29 pS. No subconductance states were seen. The current-voltage determinations for the single-channels activated by alphaxalone demonstrated a linear relationship. Mean open and shut times of single alphaxalone-activated channels were described by two exponential decay functions. Taken together, the results indicate that in embryonic human DRG neurons, micromolar concentrations of alphaxalone directly activate Cl channels whose electrophysiological and pharmacological properties are distinct from those of Cl channels associated with GABA_A receptors.